1.1 Main reagents and equipment
The Luteolin from Nanjing Susus Biotechnology Co., Ltd., the human UM cell line C918 obtained from Shanghai Fuheng Biotechnology Co., Ltd. and authenticated using short tandem repeats, fetal bovine serum (FBS) purchased from BI Biotechnology Company in Israel, RPMI 1640 culture medium from Hyclone Company in the USA, Trypsin-EDTA, streptomycin, and penicillin obtained from Wuhan Boster Biotechnology Co., Ltd., Dimethyl sulfoxide from Roche Company in Switzerland, CCK-8 kit from Beijing Quanshijin Biotechnology Co., Ltd., and a set of recombinant rabbit monoclonal antibodies, including MelanA antibody, S-100 antibody, HMB45 antibody, Ki67 antibody, and goat anti-rabbit secondary antibodies, which were sourced from Hangzhou Huaan Biotechnology Co., Ltd. Furthermore, Tris-EDTA antigen retrieval buffer, 3% hydrogen peroxide, 10% goat serum, and a DAB color development kit were obtained from Wuhan Boster Biotechnology Co., Ltd. Hematoxylin stain and Eosin stain were provided by MERCK Company. Additionally, ultra-low adsorption 96-well plates and ultra-low adsorption 6-well plates were purchased from Liwo Biotechnology Co., Ltd., while an ultra-clean workbench was sourced from Suzhou Purification Equipment Co., Ltd. The cell culture incubator and microplate reader were acquired from Thermo Scientific Company, and an inverted phase contrast optical microscope was obtained from the Japanese Olympus Company. Finally, cell culture dishes and culture plates were supplied by BIO-FIL Company.
1.2 Cell culture
C918 cells were cultured in RPMI 1640 complete medium with 10% FBS and 1% antibiotics (penicillin/streptomycin). Cells were incubated in a cell culture incubator at 37°C and 5% CO2. Once adherent, passaging occurred at a 1:6 ratio every 2 to 3 days. For three-dimensional cell culture experiments, cells were seeded at densities of 3000, 6000, and 9000 per well in round-bottom ultra-low adsorption 96-well plates to culture three-dimensional cell spheroids. Ultra-low adsorption 6-well plates were employed to culture three-dimensional multi-cell spheroids, with a 10-day incubation period. In 2D cell culture experiments, C918 cells were incubated in standard cell culture dishes and plates.
1.3 Morphological and histological analysis
Morphological changes in C918 cell spheroids were observed in real-time during three-dimensional spheroid culture using an inverted phase-contrast optical microscope and a cell imaging system. Images were captured for later analysis. After 10 days of culture, three-dimensional multicellular spheroids were prepared using histological staining methods. This involved fixing the spheroids with 4% paraformaldehyde, pre-embedding them in agarose, dehydrating the agarose with alcohol gradients, and removing residual alcohol with xylene. Subsequently, the spheroids were embedded in paraffin and sectioned. The sections were deparaffinized with xylene, rehydrated with decreasing gradient alcohol solutions, stained with hematoxylin and eosin (HE), and finally dehydrated and mounted with gum. HE staining was performed to observe and analyze the cytohistological characteristics of multicellular spheroids. To detect the expression of specific markers in multicellular spheroids, immunohistochemical staining was performed. Initially, tissue sections were heat-treated with Tris-EDTA antigen repair buffer for 20 minutes to repair cellular antigens. Next, 3% hydrogen peroxide was used to block endogenous peroxidase activity, and 10% goat serum was employed to prevent nonspecific binding. The sections were then incubated with antibodies against cell proliferation marker (Ki67) and melanoma markers (MelanA, HMB45, S-100), followed by incubation with corresponding antibodies labeled with horseradish peroxidase. Finally, DAB chromogenic solution and hematoxylin were used for counterstaining, and the slides were dehydrated and mounted. Immunohistochemical staining was conducted to identify positive expression of these markers in spheroids.
1.4 Drug treatment
Dissolve luteolin in dimethyl sulfoxide to prepare a 100 mmol/L stock solution and store it at -20℃ for later use. During the experiment, the luteolin stock solution was diluted to the desired concentration using RPMI 1640 complete medium. Luteolin concentrations of 0μmol/L, 6.25μmol/L, 12.5μmol/L, 25μmol/L, 50μmol/L, and 100μmol/L were used to treat C918 cells at a density of 6000 cells per well. The optical density at a wavelength of 450 nm was measured using the CCK-8 assay 72 hours after drug exposure. In three-dimensional cell culture, an equivalent number of cells were seeded in round-bottom ultra-low adsorption 96-well plates. Luteolin concentrations of 0μmol/L, 12.5μmol/L, 25μmol/L, 50μmol/L, 100μmol/L, 200μmol/L, and 400 μmol/L were applied for 72 hours, and the optical density at a wavelength of 450 nm was observed. For each concentration group, 6 replicate wells were set up, and the test was independently repeated three times. Based on the measurement results, the IC50 values of luteolin acting on C918 cells for 72 hours in both two-dimensional and three-dimensional cell culture models were calculated.
1.5 Cell morphology observation
Experimental groups were established based on the IC50 value of three-dimensional cultured C918 cells treated with luteolin for 72 hours. These groups included the control group (0μmol/L luteolin), the 25μmol/L luteolin group, the 50μmol/L luteolin group, the 75μmol/L luteolin group, and the 100μmol/L luteolin group. Following a 72-hour treatment with these luteolin concentrations, three-dimensional cultured C918 cells were observed using an inverted phase-contrast optical microscope to examine the cell spheroids. Each concentration group was tested with three replicate wells, and the experiments were independently repeated three times. ImageJ software was utilized to calculate the maximum cross-sectional area of the cell spheroids and record the values.
1.6 Statistical analysis
All data analysis and statistical processing were conducted using SPSS 21.0 statistical software. Two independent samples t-tests were employed for comparing two groups, while one-way ANOVA was used for comparing multiple groups. The significance level was set at 0.05.